MECHANISMS OF MEMBRANE FUSION

我们建议研究控制胞吐和胞吐的分子因素。 简化的(亚细胞)体外细胞融合事件 这些流程的模型。我们将确定个体脂质的作用 低pH和渗透压梯度对膜融合的影响及种类 通过脂质体融合分析。我们还建议确定 融合后涉及的细胞质因子和膜蛋白 含溶酶体的分离包被囊泡及其相互作用 嗜铬颗粒与嗜铬细胞质膜。个人 项目可分为以下几类： (1)个别脂类对膜融合的控制：(I)脂类 参与刺激相关的“磷脂效应”，即：三-，二-， 和单磷脂酰肌醇及其代谢副产物， 磷脂酸盐、二甘酯和花生四烯酸；(Ii)酰链 特定磷脂物种内的组合物(流动性)； 凝胶-液晶和片状-六方相变；(四) 糖脂和凝集素。 (2)低pH和渗透压梯度在膜融合中的作用：(I) 低pH(4.5-6.5)诱导的膜融合的分子需求 脂膜病毒的融合模型及其分选和分离 内吞囊泡的循环；和(Ii)渗透的贡献 脂质体融合的动力学和钙需求的梯度 具有不同的组成和大小。 (3)介导胞吐作用的细胞质和膜蛋白：作用 突触素等细胞质因子和颗粒膜蛋白 钙离子介导的嗜铬细胞颗粒与血浆的相互作用 膜。 (4)包被囊泡与囊泡融合的分子因素 溶酶体：包被的囊泡与带有荧光的溶酶体融合 检测方法及其介导的细胞质因子和膜蛋白 解包被的囊泡及其与溶酶体的融合。

We propose to study the molecular factors which control exocytotic and endocytotic fusion events by means of simplified (sub-cellular) in vitro models for these processes. We will determine the role of individual lipid species and the effects of low pH and osmotic gradients on membrane fusion by means of liposome fusion assays. We also propose to identify the cytoplasmic factors and membrane proteins involved by following the fusion of isolated coated vesicles with lysosomes and the interaction of chromaffin granules with chromaffin cell plasma membranes. The individual projects can be categorized as follows: (1) Control of membrane fusion by individual lipid species: (i) the lipids involved in the stimulus-linked "phospholipid effect, "ie: the tri-, di-, and monophosphatidylinositols and their metabolic byproducts, phosphatidate, diglycerides and arachindonic acids; (ii) acyl chain composition (fluidity) within a particular phospholipid species; (iii) the gel-liquid crystalline and lamellar-hexagonal HII phase transitions; (iv) glycolipigs and lectins. (2) The role of low pH and osmotic grandients in membrane fusion: (i) molecular requirements of low pH (4.5-6.5)-induced membrane fusion, as a model for the fusion of lipid-enveloped viruses and the sorting-out and recycling of endocytotic vesicles; and (ii) the contribution of osmotic gradients to the kinetics and Ca++ requirements of the fusion of liposomes of varying composition and size. (3) Cytoplasmic and membrane proteins which mediate exocytosis: the role synexin and other cytoplasmic factors and granule membrane proteins in the Ca++ mediated interaction of isolated chromaffin granules with plasma membranes. (4) Molecular factors involved in the fusion of coated vesicles with lysosomes: fusion of coated vesicles with lysosomes with a fluorescence assay and the cytoplasmic factors and membrane proteins which mediate the uncoating of the coated vesicles and their fusion with the lysosomes.